The invention relates to an apparatus for handling a centrifuge tube in an automatic culture system which achieves an automatic culturing of biological tissues such as cells.
As is generally recognized, the culturing of tissues such as cells takes place in a culture vessel a number of which are placed at still in a tissue culturing apparatus. In the conventional manual culturing operation of tissues, the culture vessel must be taken out of the apparatus and into the atmosphere in order to permit an observation of the results of culturing of tissues or to enable various operations which are necessary to perform a culturing over successive generations. The tissues are then moved out of a controlled environment such as a specific atmosphere, temperature and humidity maintained within the culture apparatus when it is moved into the atmosphere, and thus may be subject to a rapid change in the environment. In addition, the tissues may be contaminated by miscellaneous strains present in the atmosphere.
The present applicant has previously proposed a tissue culture apparatus which enables all of necessary culturing operations to be automatically performed under a given environment maintained in the interior thereof in order to overcome above difficulties. With this automatic culture apparatus, tissues which are to be cultured over successive generations are diluted with a culture solution and are injected into a culture vessel in suspension, and the vessel is placed at still under a specific environment maintained within the culture apparatus for purpose of culturing. After a given period of time, the vessel is conveyed to an observation position within the apparatus for inspection of the culturing result under a microscope. When it is confirmed that the tissue growth has increased to the full extent of the vessel, it is moved to a distribution position within the apparatus where a culturing solution is drawn with a pipette from the vessel and disposed. Subsequently, the tissue remaining in the vessel is rinsed by injection of a buffer solution, which is then drawn with the pipette and disposed. Subsequently, an enzyme solution such as tripsin is injected into the vessel and the latter allowed to stand still until the tissues which attached to the bottom surface of the vessel is almost free to be released therefrom. Thereafter, the enzyme solution is drawn with the pipette from the vessel and disposed, and a culture solution is again injected into the culture vessel. By repeatedly withdrawing and discharging the culture solution with the pipette, the solution is agitated to free the tissue completely from the bottom surface of the vessel, leaving it in suspension in the culture solution. The tissues in suspension are removed with the pipette and transferred into a centrifuge tube, which is subjected to the action of a centrifuge in order to achieve a separation between the culture solution and the tissues. Thereupon, the tissues having a greater specific gravity will attach to the bottom of the tube while the culture solution will be supernatant. The unnecessary culture solution is disposed by decantering the centrifuge tube. Subsequently, a culture solution is again injected into the tube, and is also agitated by the similar withdrawal and discharge operation mentioned above, using a pipette, so that the tissues are in uniform suspension in the culture solution contained within the tube. It is then distributed into a pair of fresh culture vessels in equal amounts, thus completing a single culturing operation. The culturing of tissues over successive generations requires a plurality of such culturing operations to be effected upon respective tissues contained in a number of culture vessels.
The centrifugation is required during the culturing operation because the enzyme solution which is injected into the vessel to render the tissues, which adhere to the bottom surface thereof, almost free cannot be completely withdrawn and disposed by the use of a pipette. A small amount of the enzyme solution may remain adhering to the bottom surface of the vessel or between the tissues. Under this condition, the tissues cannot be suspended in the fresh culture solution which is injected subsequently when they are distributed into the pair of fresh culture vessels. For this reason, the centrifugation is utilized while the tissues are in suspension in the previous culture solution in order to separate any remaining enzyme solution from the tissues. Also, part of the tissues which deceased in the process of culturing is also separated from the tissues which are to be used for the distribution.
To perform the various operations which are necessary to achieve the distribution from each of a number of culture dishes efficiently in the automatic culture apparatus described, it may be contemplated to provide a turntable on which a number of centrifuge tubes are mounted so that various operations can be performed at each angular position of the turntable. A centrifuge tube is mounted on the turntable at one angular position thereof, but must then be transferred to or from the rotor of a centrifuge, which constitutes a separate rotational system from the turntable. During the transfer, the rotor must be maintained stationary. However, the length of time during which the rotor is maintained stationary must be reduced as short as possible, since the total number of tissue specimens to be treated is almost doubled from generation to generation, so that toward the end of the culturing over successive generations, it will become necessary to operate the centrifuge almost continuously. Thus, the length of time during which the rotor must be maintained stationary represents a limitation on the number of generations over which the culturing can be continued.
The automatic culture apparatus described is provided with a distributor which is adapted to receive a pipette, falling in a vertical position, and which is rotatable to convey or transfer the pipette between a culture vessel and a centrifuge tube in various steps such as the withdrawal and disposal of the various solutions from the culture vessel, the transfer of the grown cells maintained in suspension in the culture solution into the centrifuge tube subsequent to the agitation of the solution in the vessel, and the distribution into a pair of fresh culture vessels of grown cells maintained in suspension in the centrifuge tube after the agitation thereof and the centrifugation. It is necessary that the pipette be replaced by a fresh one upon completion of each step mentioned above. Otherwise the solution of the previous step which adheres to the pipette may be admixed into liquid contained in a fresh culture vessel or centrifuge tube which is supplied to perform the next following step, thus exerting a significant adverse influence upon the culturing operation. For this reason, it is necessary to provide a very large number of sterilized pipettes during the operation of the automatic culture apparatus, and to feed them to the distributor or pipette supply station in synchronism with the operation of the apparatus.
For the same reasons, the centrifuge tube which is used for separation of the grown tissue or cells from unnecessary culture solution must be replaced for each specimen in order to prevent the contamination between successive specimens. As a consequence, a number of centrifuge tubes must be supplied to the automatic culture apparatus in order to permit a culturing of a number of tissue specimens. It is necessary to note that the centrifuge tube must be supplied one by one. A conventional apparatus employs a chain drive to feed the tube one by one. However, this arrangement does not lend itself to the culturing over successive generations since separate tubes must be supplied in continuous succession and since they cannot be readily sterilized.
The requirements for an arrangement which supplies a centrifuge tube to the automatic culture apparatus are given below.
1. The limited space allowance within an incubator which contains a variety of mechanisms and the number of centrifuge tubes required necessitate the capability to supply centrifuge tubes from the exterior of the incubator so that they can be additionally supplied whenever required.
2. The internal environment or atmosphere of the incubator must not be disturbed when externally supplying the centrifuge tube since a delicate control is involved.
3. Provision must be provided to facilitate the sterilization in order to prevent the penetration of the miscellaneous strains into the incubator.
4. Reliability of supplying centrifuge tubes one by one.
5. The operation and handling can be easily achieved.
As mentioned above, during the culturing process in the automatic culture apparatus, cells which have grown sufficiently within the vessel are injected into a centrifuge tube by using a pipette, and the tube subjected to centrifugation to separate the cells from the supernatant solution and deceased cells. Finally, the supernatant solution is disposed, and the empty centrifuge tube is disposed after completion of the distributing operation. The disposal of the centrifuge tube after its use is necessary because the cells cultured within the centrifuge tube are very susceptible to contamination by miscellaneous strains. Therefore, it is desirable that the centrifuge tube to which a supernatant solution from other cells being cultured may have attached be replaced by a sterilized centrifuge tube in order to minimize the attachment and growth of the miscellaneous strains and to assure the uniform quality of the tissues being cultured.